In view of wide range of biological properties associated with 1,4-DHP and owing to the biological importance of the oxidation step of 1,4-DHP, we carried out the synthesis and antimicro
Trang 1O R I G I N A L Open Access
Synthesis and antimicrobial evaluation of new
1,dihydro-pyrazolylpyridines and
4-pyrazolylpyridines
Om Prakash1, Khalid Hussain2, Ravi Kumar3*, Deepak Wadhwa4, Chetan Sharma5and Kamal R Aneja5
Abstract
Background: Dialkyl 1,4-dihydro-2,6-dimethylpyridine-3,5-dicarboxylates (1,4-DHP) have now been recognized as vital drugs Some of these derivatives such as amlodipine, felodipine, isradipine, etc have been commercialized In view of wide range of biological properties associated with 1,4-DHP and owing to the biological importance of the oxidation step of 1,4-DHP, we carried out the synthesis and antimicrobial evaluation of new diethyl 1,4-dihydro-4-pyrazolyl)pyridine-3,5-dicarboxylates (2a-g) and diethyl 2,6-dimethyl-4-(3-aryl-1-phenyl-4-pyrazolyl)pyridine-3,5-dicarboxylates (3a-g)
Results: Synthesis of a series of new diethyl 1,4-dihydro-2,6-dimethyl-4-(3-aryl-1-phenyl-4-pyrazolyl)pyridine-3,5-dicarboxylates (2a-g) has been accomplished by multicomponent cyclocondensation reaction of ethyl
acetoacetate, 3-aryl-1-phenyl pyrazole-4-carboxaldehyde (1a-g) and ammonium acetate The dihydropyridines 2a-g were smoothly converted to new diethyl 2,6-dimethyl-4-(3-aryl-1-phenyl-4-pyrazolyl)pyridine-3,5-dicarboxylates (3a-g) using HTIB ([Hydroxy (tosyloxy)iodo]benzene, Koser’s reagent) as the oxidizing agent The antimicrobial studies
of the title compounds, 2a-g &3a-g, are also described
Keywords: 1,4-Dihydro-4-pyrazolylpyridines, 4-pyrazolylpyridines, HTIB, oxidation, antibacterial activity, antifungal activity
Background
Dialkyl
1,4-dihydro-2,6-dimethylpyridine-3,5-dicarboxy-lates (1,4-DHP; Figure 1) have now been recognized as
vital drugs Some of these derivatives, such as amlodipine,
felodipine, isradipine, etc have been commercialized, and
it has been proven that their therapeutic success is
related to their efficacy to bind to calcium channels and
consequently to decrease the passage of the
transmem-brane calcium current [1-3] Further, cerebrocrast, a
dihydropyridine derivative, has been introduced as a
neu-roprotective agent [4] Together with calcium channel
blocker and neuroprotective activity, a number of
dihy-dropyridine derivatives have been found as vasodilators,
antihypertensive, bronchodilators, antiatherosclerotic,
hepatoprotective, antitumour, antimutagenic,
geroprotec-tive, antidiabetic and antiplatelet aggregation agents
[5-9] In a recent article, 4-[5-chloro-3-methyl-1-phenyl-1H-pyrazol-4-yl]-dihydropyridines have been shown to possess significant antimicrobial activity [10]
In addition to above, aromatization of 1,4-DHP has also attracted considerable attention in recent years as Böcker has demonstrated that metabolism of the above drugs involves a cytochrome P-450 catalysed oxidation
in the liver [11]
In view of wide range of biological properties asso-ciated with 1,4-DHP and the biological importance of the oxidation step of 1,4-DHP, we carried out the synthesis and antimicrobial evaluation of new diethyl 1,4-dihydro- 1-phenyl-4-pyrazolyl)pyridine-3,5-dicarboxylates (2a-g) and diethyl 2,6-dimethyl-4-(3-aryl-1-phenyl-4-pyrazolyl)pyridine-3,5-dicarboxylates (3a-g)
Results and discussion
Chemistry
The synthetic scheme used for the synthesis of diethyl 1,4-dihydro-2,6-dimethyl-4-(3-aryl-1-phenyl-4-pyrazolyl)
* Correspondence: ravi.dhamija@rediffmail.com
3 Department of Chemistry, Dyal Singh College, Karnal 132 001, India
Full list of author information is available at the end of the article
© 2011 Prakash et al; licensee Springer This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium,
Trang 2pyridine-3,5-dicarboxylates (2a-g) is outlined in Scheme
1 Synthesis of the title compounds 2a-g was
accom-plished by multicomponent cyclocondensation reaction
of ethyl acetoacetate,
3-aryl-1-phenyl-pyrazole-carboxal-dehyde (1a-g) and ammonium acetate in ethanol The
purity of the compounds was checked by TLC and
ele-mental analysis Spectral data (IR,1H NMR (see
addi-tional files 1, 2, 3, 4 and 5, mass) of the newly
synthesized compounds 2a-g were in full agreement
with their proposed structures The IR spectra of
com-pounds 2a-g exhibited characteristic peak at
approxi-mately 1697 cm-1 because of the presence of ester group
(-COOEt), and peak due to -N-H stretch appeared in
the region 3300-3317 cm-1 In1H NMR of compounds
2a-g, the protons of C4-H and -NH of the
dihydropyri-dine ring resonate betweenδ 5 and 6 ppm
Hypervalent iodine (III) and iodine (V) reagents have
been used as green-oxidants for a variety of substrates
[12-17] Amongst the various reagents used, HTIB has
been reported to serve as a mild, fast and efficient
oxi-dant for the aromatization of Hantzsch
1,4-dihydropyri-dines to pyri1,4-dihydropyri-dines [18]
Thus, diethyl 1,4-dihydro-2,6-dimethyl-4-(3-aryl-1-phenyl-4-pyrazolyl) pyridine-3,5-dicarboxylates (2a-g) were further oxidized by treating with HTIB (Koser’s reagent) in dichloromethane (CH2Cl2) at room tempera-ture to afford new diethyl 2,6-dimethyl-4-(3-aryl-1-phe-nyl-4-pyrazolyl) pyridine-3,5-dicarboxylates (3a-g) in good-to-excellent yields (Scheme 1) All the compounds 3a-g were unambiguously characterized on the basis of their spectral (IR,1H NMR (see additional files 6, 7, 8,
9, 10, 11 and 12) and mass) and elemental data
A plausible mechanism for the oxidation of dihydro-pyridines 2 to 3 is outlined in Scheme 2 The probable mechanism might involve the attack by N-H on PhI (OH)OTs, leading to the formation of intermediate 4 The intermediate 4 finally loses a molecule of iodoben-zene (PhI) to give 3
Pharmacology
All the synthesized compounds, 2a-g and 3a-g, were evaluated in vitro for their antibacterial activity against two gram-positive bacterial strains, Staphylococcus aur-eus &Bacillus subtilis and two gram-negative bacteria, namely, Escherichia coli and Pseudomonas aeruginosa and their activities were compared with a well-known commercial antibiotic, ciprofloxacin In addition, the synthesized compounds were also evaluated for their antifungal activity against Aspergillus niger &Aspergillus flavus and their antifungal potential was compared to reference drug, fluconazole Compounds possessed vari-able antibacterial activities against Gram-positive bac-teria, S aureus, B subtilis However, the compounds in this series were not effective against any Gram-negative bacteria, neither against E coli nor against P aeruginosa Results of antibacterial evaluation are summarized in Table 1
Compounds 2a-g and 3a-g showed zones of inhibition ranging between 14 and 20 mm On the basis of the zones of inhibition produced against the test bacteria, compounds 2b and 3a were found to be most effective against S aureus, showing the maximum zones of inhi-bition at 18 and 20 mm, respectively, and compounds 3a, 3e and 3g were found to be most effective against B
Figure 1 1,4-DHP.
Scheme 1 Synthesis of 1,4-DHP (2) and aromatization of 2 to 3
using HTIB. Scheme 2 Proposed mechanism for the oxidation of 2 to 3.
Trang 3subtilis The remaining compounds showed fair activity
against gram-positive bacterial strains (Table 1) In the
whole series, the MIC (minimum inhibitoty
concentra-tion) values of various tested chemical compounds
ran-ged between 64 and 256μg/mL against gram-positive
bacteria Compounds 2b and 3a displayed good
antibac-terial activity with the lowest MIC value, 64 μg/ml
against S aureus Three compounds, 3a, 3e and 3g
pos-sessed antibacterial activity with MIC value of 64μg/mL
against B subtilis (Table 2)
Amongst the synthesized compounds, six compounds
2a, 2d, 2g, 3a, 3c and 3d showed more than 50%
myce-lial growth inhibition against A niger whereas
com-pounds, 2a, 2e, 2f, 3a, 3d and 3f were found to be
active against A flavus (Table 3)
From the overall result it is evident that compound 3a
could be identified as the most biologically active
member within this study with good antifungal and anti-bacterial profile
Conclusions
A series of diethyl 1,4-dihydro-2,6-dimethyl-4-(3-aryl-1-phenyl-4-pyrazolyl)pyridine-3,5-dicarboxylates (2a-g) and diethyl 2,6-dimethyl-4-(3-aryl-1-phenyl-4-pyrazolyl)pyri-dine-3,5-dicarboxylates (3a-g) has been synthesized with the hope of discovering new structure leads Compounds 2band 3a were found to be most effective against S aur-eusshowing the maximum zones of inhibition of 18 and
20 mm, respectively, and compounds 3a, 3e and 3g were found to be most effective against B subtilis Moreover, six compounds 2a, 2d, 2g, 3a, 3c and 3d showed more than 50% mycelial growth inhibition against A niger whereas compounds, 2a, 2e, 2f, 3a, 3d and 3f were found
to be active against A flavus; however, no compound was found superior over the reference drug
Finally, compound 3a could be identified as the most biologically active member within this study with an interesting antibacterial and antifungal profile
Experimental
Chemical synthesis
Melting points were taken in open capillaries and are uncorrected IR spectra were recorded on Perkin-Elmer
IR spectrophotometer The 1H NMR spectra were recorded on Brucker 300 MHz instrument The chemi-cal shifts are expressed in ppm units downfield from an internal TMS standard 3-Aryl-1-phenylpyrazole-4-car-boxaldehydes (1a-h), needed for the present study, were synthesized by Vilsmeier-Haack reaction according to the literature procedure [19]
Synthesis of diethyl 1,4-dihydro-2,6-dimethyl-4-(3-aryl-1-phenyl-4-pyrazolyl) pyridine-3,5-dicarboxylates (2a-g)
General procedure: A mixture of appropriate 3-aryl-1-phenylpyrazole-4-carboxaldehyde (1, 10 mmol), ethyl acetoacetate (20 mmol) and ammonium acetate (22 mmol) in ethanol was allowed to reflux on water bath for 25-30 min After completion of the reaction, the
Table 1 Antibacterial activity of chemical compounds
through agar well diffusion method
Compound Diameter of growth of inhibition zone (mm)a
S aureus Bacillus Subtilis E coli P aeruginosa
-Ciprofloxacin 27.6 26.3 25.0 25.3
-, No activity
a
Values, including diameter of the well (8 mm), are means of three replicates
Table 2 MIC (inμg/mL) of compounds obtained using
macrodilution method
Compound S.
aureus
Bacillus Subtilis
Compound S.
aureus
Bacillus Subtilis
Ciprofloxacin 5 5
Table 3 Antifungal activity of chemical compounds through poisoned food method (mycelial growth inhibition) (%)
Compound A niger A flavus Compound A niger A flavus
Fluconazole 81.1 77.7
Trang 4reaction mixture was cooled to room temperature to
give pure diethyl
1,4-dihydro-2,6-dimethyl-4-(3-aryl-1-phenyl-4-pyrazolyl) pyridine-3,5-dicarboxylates (2a-g)
Characterization data of diethyl
1,4-dihydro-2,6-dimethyl-4-(3-aryl-1-phenyl-4-pyrazolyl) pyridine-3,5-dicarboxylates (2a-g)
2a: M.p.: 124°C; yield: 74%; IR (νmax, cm-1, KBr): 3323
(NH stretch), 1690 (-COOEt), 1207;1H NMR (CDCl3,δ,
ppm): 1.069-1.115 (t, 6H), 2.237 (s, 6H), 3.744-4.068 (m,
4 H), 5.318 (s, 1 H), 5.544 (s, 1 H), 7.221-7.424 (m, 4
H), 7.806 (s, 1 H), 7.681-7.868 (m, 6 H); mass: m/z
472.30 (M++ 1, 100%)
Anal Calcd for C28H29N3O4: C 71.33, H 6.15, N 8.91;
found: C 71.34, H 6.18, N 8.94; C 71.33, H 6.15, N 8.91
2b: M.p.: 189°C; yield: 70%; IR (νmax, cm-1, KBr): 3325
(NH stretch), 1697 (-OOEt), 1643, 1211; 1HNMR
(CDCl3, δ, ppm): 1.032-1.087 (t, 6 H), 2.225 (s, 6 H),
2.401 (s, 3 H), 3.730-4.095 (m, 4 H), 5.306 (s, 1 H),
5.722 (bs, 1 H), 7.205-7.282 (m, 3 H), 7.381-7.450 (m, 2
H), 7.664-7.692 (m, 2 H), 7.733 (s, 1 H), 7.742-7.769 (d,
2 H, J = 8.1 Hz); mass: m/z 486.20 (M++ 1, 100%)
Anal Calcd for C29H31N3O4: C 71.75, H 6.39, N 8.66;
found: C 71.71, H 6.42, N 8.66
2c: M.p.: 139°C; yield: 78%; IR (νmax, cm-1, KBr): 3317
(NH stretch), 1697 (-COOEt), 1643, 1211; 1H NMR
(CDCl3, δ, ppm): 1.079-1.127 (t, 6 H), 2.250 (s, 6 H),
3.866 (s, 3 H), 3.801-4.102 (m, 4 H), 5.288 (s, 1 H),
5.561 (s, 1 H), 6.962-6.991 (d, 2 H, J = 8.7 Hz),
7.209-7.440 (m, 3 H), 7.670-7.697 (d, 2 H, J = 8.7 Hz) 7.742 (s,
1 H), 7.785-7.814 (d, 2 H, J = 8.7 Hz); mass: m/z 502.32
(M+ + 1, 100%)
Anal Calcd for C29H31N3O5: C 69.46, H 6.19, N 8.38;
found: C 69.42, H 6.24, N 8.37
2d: M.p.: 175°C; yield: 72%; IR (νmax, cm-1, KBr): 3333
(NH stretch), 1697 (-COOEt), 1643, 1211; 1H NMR
(CDCl3, δ, ppm): 0.940-0.975 (t, 6 H), 2.521 (s, 6 H),
4.102-4.132 (m, 4 H), 5.175 (s, 1 H), 5.562 (s, 1 H),
6.962-6.991 (d, 2 H, J = 8.7 Hz), 7.281-7.513 (m, 5 H),
7.734 (d, 2 H, J = 7.5 Hz), 7.922 (s, 1 H); mass: m/z
490.26 (M++ 1, 100%)
Anal Calcd for C28H28N3O4F: C 68.71, H 5.73, N
8.58; found: C 68.72, H 5.75, N 8.56
2e: M.p.: 185°C; Yield: 76%; IR (νmax, cm-1, KBr): 3317
(NH stretch), 1697 (-COOEt), 1636, 1211; 1H NMR
(CDCl3, δ, ppm): 1.072-1.119 (t, 6 H), 2.280 (s, 6 H),
3.790-4.080 (m, 4 H), 5.285 (s, 1 H), 5.551 (s, 1 H),
7.235-7.454 (m, 5 H), 7.668-7.694 (d, 2 H) 7.814 (s, 1
H), 7.863-7.891 (d, 2 H, J = 8.4 Hz); mass: m/z 506.26,
508.24
Anal Calcd for C28H28N3O4Cl: C 66.47, H 5.54, N
8.31; found: C 66.47, H 5.55, N 8.31
2f: M.p.: 174°C; yield: 72%; IR (νmax, cm-1, KBr): 3564
(NH stretch), 1728 (-COOEt), 1242;1H NMR (CDCl3,δ,
ppm): 1.072-1.119 (t, 6 H), 2.275 (s, 6 H), 3.764-4.104
(m, 4 H), 5.284 (s, 1 H), 5.581 (s, 1 H), 7.234-7.452 (m,
3 H), 7.561-7.588 (d, 2 H, J = 7.8 Hz), 7.665-7.691 (d, 2
H, J = 7.8 Hz) 7.753 (s, 1 H), 7.806-7.834 (d, 2 H, J = 8.4 Hz); mass: m/z 550.31, 552.31
Anal Calcd for C28H28N3O4Br: C 61.20, H 5.10, N 7.65; found: C 61.09, H 5.14, N 7.64
2g: M.p.: 198°C; yield: 70%; IR (νmax, cm-1, KBr): 3302 (NH stretch), 1697 (-COOEt), 1636, 1211; 1H NMR (CDCl3, δ, ppm): 1.026-1.071 (t, 6 H), 2.325 (s, 6 H), 3.775-4.047 (m, 4 H), 5.335 (s, 1 H), 5.766 (s, 1 H), 7.282-7.473 (m, 4 H), 7.684-7.709 (d, 2 H, J = 7.5 Hz), 7.801 (s, 1 H), 8.254-8.344 (m, 3 H); mass: m/z 517.29 (M+ + 1, 100%)
Anal Calcd for C28H28N4O6: C 65.11, H 5.42, N 10.85; found: C 65.13, H 5.47, N 10.83
Synthesis of diethyl 2,6-dimethyl-4-(3-aryl-1-phenyl-4-pyrazolyl)pyridine-3,5-dicarboxylates (3a-g)
General procedure: To a solution of appropriate 1,4-DHP (2, 10 mmol) in dichloromethane, was added HTIB (12 mmol) and the mixture was stirred at room temperature The progress of the reaction was moni-tored by TLC Reaction was completed in 4-5 min After the completion of reaction, the reaction mixture was washed with aqueous NaHCO3 solution Organic phase was then separated, dried and concentrated on water bath Crude product, thus obtained, was purified
by silica gel column chromatography using Pet ether/ EtOAc (20:1) as eluent to afford pure diethyl 2,6- dimethyl-4-(3-aryl-1-phenyl-4-pyrazolyl)pyridine-3,5-dicarboxylates (3a-g)
Characterization data of dimethyl 2,6-dimethyl-4-pyrazolylpyridine-3,5-dicarb oxylates (3a-g)
3a: M.p.: 111°C; yield: 68%; IR (νmax, cm-1, KBr): 1736, 1233; 1H NMR (CDCl3, δ, ppm): 0.911-0.997 (t, 6 H), 2.613 (s, 6 H), 3.910-4.07 (m, 4 H), 7.110-7.313 (m, 4 H), 7.817 (s, 1 H), 7.581-7.690 (m, 6 H); mass: m/z 470.20 (M++ 1, 100%)
Anal Calcd for C28H27N3O4: C 71.64, H 5.76, N 8.95; found: C 71.63, H 5.79, N 8.93
3b: M.p.: 105°C; yield: 69%; IR (νmax, cm-1, KBr): 1720, 1234; 1H NMR (CDCl3, δ, ppm): 0.913-0.960 (t, 6 H), 2.611 (s, 6 H), 2.468 (s, 3H), 3.923-4.072 (q, 4 H), 6.839-6.868 (d, 2H, J=8.7 Hz), 7.280-7.501 (m, 5 H), 7.732-7.759 (d, 2 H, J = 8.7 Hz), 7.905 (s, 1 H); mass: m/z 484.40 (M++ 1, 100%)
Anal Calcd for C29H29N3O4: C 72.05, H 6.00, N 8.70; found: C 72.06, H 6.05, N 8.70
3c: M.p.: 136°C; Yield- 72%; IR (νmax, cm-1, KBr): 1740, 1034; 1H NMR (CDCl3, δ, ppm): 0.913-0.998 (t, 6 H), 2.612 (s, 6 H), 3.808 (s, 3 H), 3.924-4.08 (q, 4 H), 6.835-6.864 (d, 2 H, J = 8.7 Hz), 7.311-7.501 (m, 5 H), 7.732-7.759 (d, 2 H, J = 8.7 Hz), 7.905 (s, 1 H); mass: m/z 500.29 (M++ 1, 100%)
Anal Calcd for C29H29N3O5: C 69.73, H 5.81, N 8.41; found: C 69.71, H 5.83, N 8.40
Trang 53d: M.p.: 121°C; yield: 70%; IR (νmax, cm-1, KBr): 1728,
1236, 1037;1H NMR (CDCl3, δ, ppm): 0.924-0.971 (t, 6
H), 2.615 (s, 6 H), 3.905-4.105 (q, 4 H), 6.987-7.044 (m,
2 H), 7.280-7.365 (m, 1 H), 7.469-7.622 (m, 4 H),
7.733-7.759 (d, 2 H, J = 7.8 Hz), 7.923 (s, 1 H); mass: m/z
488.36 (M++ 1, 100%)
Anal Calcd for C28H26N3O4F: C 68.99, H 5.38, N
8.62; found: C 68.95, H 5.37, N 8.63
3e: M.p.: 101-102°C, lit [20] M.p.: 101-102°C; Yield:
65%
3f: M.p.: 115°C; yield: 70%; IR (νmax, cm-1, KBr): 1734,
1030; 1H NMR (CDCl3, δ, ppm): 0.940-0.962 (t, 6 H),
2.617 (s, 6 H), 3.957-4.039 (q, 4 H), 7.200-7.495 (m, 7
H), 7.732-7.756 (d, 2 H, J = 7.2 Hz), 7.921 (s, 1 H);
mass: m/z 548.20, 550.20
Anal Calcd for C28H26N3O4Br: C 61.42, H 4.75, N
7.68; found: C 61.31, H 4.79, N 7.69
3g: M.p.: 172°C; yield: 68%; IR (νmax, cm-1, KBr): 1728,
1234, 1034;1H NMR (CDCl3, δ, ppm): 0.895-0.941 (t, 6
H), 2.632 (s, 6 H), 3.923-4.039 (m, 4 H), 7.279-7.410 (m,
3 H), 7.499-7.769 (m, 4 H), 7.960 (s, 1 H), 8.178-8.207
(d, 2 H, J = 7.5 Hz); mass: m/z 515.26 (M++ 1, 100%)
Anal Calcd for C28H26N4O6: C 64.37, H 4.98, N 10.73;
found: C 65.34, H 5.08, N 10.87
Pharmacology
Test microorganisms
Total six microbial strains were selected on the basis of
their clinical importance in causing diseases in humans
Two Gram-positive bacteria (S aureus MTCC 96 and B
subtilisMTCC 121); two Gram-negative bacteria (E coli
MTCC 1652 and P aeruginosa MTCC 741) and two
fungi (A niger and A flavus) the ear pathogens isolated
from the patients of Kurukshetra [21], were used in the
present study for the evaluation of antimicrobial
activ-ities of the chemical compounds All the cultures were
procured from Microbial Type Culture Collection
(MTCC), IMTECH, Chandigarh The bacteria and fungi
were subcultured on Nutrient agar and Sabouraud’s
dex-trose agar (SDA), respectively, and incubated aerobically
at 37°C
In vitro antibacterial activity
The antibacterial activities of compounds, 2a-g and
3a-g, were evaluated by the agar well diffusion method All
the cultures were adjusted to 0.5 McFarland standard,
which is visually comparable to a microbial suspension
of approximately 1.5 × 108 cfu/mL 20 mL of Mueller
Hinton agar medium was poured into each Petri plate,
and the agar plates were swabbed with 100 μL inocula
of each test bacterium and kept for 15 min for
adsorp-tion Using sterile cork borer of 8-mm diameter, wells
were bored into the seeded agar plates, and these were
then loaded with a 100μL volume with concentration of 2.0 mg/mL of each compound reconstituted in the dimethylsulphoxide (DMSO) All the plates were incu-bated at 37°C for 24 h Antibacterial activity of each compound was evaluated by measuring the zone of growth inhibition against the test organisms with zone reader (Hi Antibiotic zone scale) DMSO was used as a negative control whereas ciprofloxacin was used as a positive control This procedure was performed in three replicate plates for each organism [22,23]
Determination of minimum inhibitory concentration
Minimum inhibitory concentration (MIC) is the lowest concentration of an antimicrobial compound that will inhibit the visible growth of a microorganism after over-night incubation MIC of the compounds against bacter-ial strains was tested through a macrodilution tube method as recommended by NCCLS [24] In this method, various test concentrations of chemically synthesized compounds were made from 256 to 1 μg/
mL in sterile tubes, 1-10 100 μL sterile Mueller Hinton Broth was poured in each sterile tube, and followed by addition of 200 μL test compound in tube 1 Twofold serial dilutions were carried out from tubes 1 to 10, and excess broth (100 μL) was discarded from the tube 10
To each tube, 100 μL of standard inoculum (1.5 × 108
cfu/mL) was added Ciprofloxacin was used as control Turbidity was observed after incubating the inoculated tubes at 37°C for 24 h
In vitro antifungal activity
The antifungal activity of the synthesized chemical com-pounds was evaluated by poison food technique The moulds were grown on SDA at 25°C for 7 days and used as inocula 15 mL of molten SDA (45°C) was poi-soned by the addition of 100 μL volume of each com-pound having concentration of 4.0 mg/mL, reconstituted in the DMSO, poured into a sterile Petri plate and allowed to solidify at room temperature The solidified poisoned agar plates were inoculated at the centre with fungal plugs (8-mm diameter), obtained from the actively growing colony and incubated at 25°C for 7 days DMSO was used as the negative control whereas fluconazole was used as the positive control The experiments were performed in triplicates Dia-meter of the fungal colonies was measured and expressed as percent mycelial inhibition determined by applying the following formula [25]:
Inhibition of mycelial growth % =(dc − dt) /dc × 100
where dc is the average diameter of fungal colony in negative control plates, and dt the average diameter of fungal colony in experimental plates
Trang 6Additional material
Additional file 1: 1HNMR spectrum of compound 2b.
Additional file 2: 1HNMR spectrum of compound 2c.
Additional file 3: 1HNMR spectrum of compound 2e.
Additional file 4: 1HNMR spectrum of compound 2f.
Additional file 5: 1HNMR spectrum of compound 2g.
Additional file 6: 1HNMR spectrum of compound 3a.
Additional file 7: 1HNMR spectrum of compound 3b.
Additional file 8: 1HNMR spectrum of compound 3c.
Additional file 9: 1HNMR spectrum of compound 3d.
Additional file 10: 1HNMR spectrum of compound 3e.
Additional file 11: 1HNMR spectrum of compound 3f.
Additional file 12: 1HNMR spectrum of compound 3g.
Abbreviations
1,4-DHP: dialkyl 1,4-dihydro-2,6-dimethylpyridine-3,5-dicarboxylates; DMSO:
dimethylsulphoxide; HTIB: hydroxy (tosyloxy)iodobenzene; MIC: minimum
inhibitory concentration; MTCC: microbial type culture collection; SDA:
Sabouraud dextrose agar.
Acknowledgements
We are thankful to the CSIR, New Delhi (Grant no CSIR 01 (2816)/07/EMR-II)
for providing financial assistance to accomplish this research The authors
are also grateful to the CSIR for the award of junior research fellowship to
Khalid Hussain.
Author details
1
Institute of Pharmaceutical Sciences, Kurukshetra University, Kurukshetra 136
119, India 2 Department of Chemistry, Guru Nanak Khalsa College,
Yamunanagar 135001, India 3 Department of Chemistry, Dyal Singh College,
Karnal 132 001, India4Department of Chemistry, Kurukshetra University,
Kurukshetra 136 119, India 5 Department of Microbiology, Kurukshetra
University, Kurukshetra 136 119, India
Competing interests
The authors declare that they have no competing interests.
Received: 21 March 2011 Accepted: 3 August 2011
Published: 3 August 2011
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